Research in the Molecular Pathogenesis is focused on defining changes in the genes that underlie inherited susceptibilities to common diseases such as cancer and birth defects. Changes in folate metabolism are correlated tumor formation and birth defects. Folate genes are also involved in the methylation of DNA and proper brain function. . We are searching for genetic variants in genes related to folate, methionine and homocysteine metabolism. Individuals affected with cancer or Spina Bifida (one form of neural tube defects) will be tested for these variants. Variants found at higher frequency in individuals with disease will help us identify genes associated with risk. In the past we found that variants in one of these genes, TC2, appear to affect the levels of vitamin B12 in the blood during pregnancy. This finding may be related to birth defects and also may help to explain why some elderly individuals become anemic and suffer neurological symptoms from vitamin B12 deficiency. We also found that mothers carrying a specific variant in a second gene, MTHFD1, have a 50% increased risk bearing a child with a neural tube defect. This previously un-described variant may be responsible for up to 25% of all neural tube defects. Approximately one in five individuals in the population carry one of these risk factors. We recently determined that this particular variant was also an risk factor for placental abruption a common cause of miscarriage and for misscarriages that occur in the second trimester. We have re-created these genes in the laboratory and are currently using an experimental system to determine exactly how these variants alter the function of these proteins. Early in 2008 we have tested more than 64 additional genes for variants that might perturb folate metabolism and therefore be associated with an increase risk of having a child with an neural tube defect. This was carried out by genotyping more than 1,200 single nucleotide polymorphisms in a large number of families affected with neural tube defects and unaffected controls. This large experiment has allowed us to exclude most of the genes on this list. Results for approximately a dozen genes suggest that they are associated with neural tube defects. Over the past year we have carried out second series of experiments to determine if the genes identified in the first stage of these are definitively associated with neural tube defects. These data are currently being analyzed. One of these genes has already passed through our stage 2 validation. This gene produces a protein that binds vitamin B12 and transports from the blood into the tissues. Our new data data that suggest that several variants in this transporter are associated with a risk of having a child with an NTD. While we now know which variants are associated with risk, we do not yet know if they are actually causing the risk or are linked to additional variants that change the function of the protein. To screen for additional variants, we sequenced the DNA containing this transporter gene in a large number of individuals. This sequencing experiment uncovered an number of previously unidentified variants in this gene. We are measured the impact of these variants on the function of the transporter. The majority variants tested do not appear to have an adverse effect of the function of the receptor. However, we have found that several of the variants are associated with changes in vitamin B12 levels. We are now testing to see i how these variants changes vitamin B12 levels and if these changes are associated with human disease conditions. These same variants do impact metabolic levels of vitamin B12 as large sample of healthy young individuals. When we perform the same test in pregnant women, the variant is associated with decreased levels of vitamin B12. This result is consistent with the association of the same variant and adverse outcomes in pregnancy. We have also carried out experiment aimed at determining the relationship between folate, vitamin B12 and DNA methylation. Little is known about inter-individual variation in DNA methylation. We developed an assay that allows us to measurethe extent of methylation by carrying out DNA sequencing. We used this assay on a large number of unrelated individuals and discovered that there is a wide range of methylation in healthy individuals. As part of this process, we have mapped DNA methylation sites in the genome at high resolution. Preliminary data indicates that variation in the MTHFR gene is not associated with changes in site-specific methylation. A detailed knowledge of the function of the the genes in the folatevitamin B12 metabolic pathways willadd to our understanding of neural tube defects and potentially help guide public health policy in the area of nutritional supplementation.